Currently only two well characterized enzymatic systems are known to catalyze the formation of an O-O bond as their primary function. They are the heme-containing chlorite dismutases (Cld) found in the perchlorate respiratory pathway of several Proteobacteria and the oxygen-evolving complex of photosystem II. Clds degrade toxic chlorite by converting it to O2 and Cl-. The rarity of the O-O bond-forming reaction of Clds and their utility to detoxify chlorite or to produce O2 on demand in a variety of biomedical and technical applications resulted in considerable interest in these enzymes. Turns out, Clds comprise a large, widespread family of enzymes that, despite their common structural fold, have varied functions. One of our long term goals is to understand how subtle differences in the heme-protein interactions elicit the varied, and in some cases unique, functions of this family. Here we propose to study representative Clds from three types within the family: Dechloromonas aromatica Cld (DaCld) which produces O2 from chlorite with tremendous efficiency for detoxification of perchlorate reduction products during anaerobic respiration; Klebsiella pneumoniae Cld (KpCld) which catalyzes the chlorite decomposition reaction less efficiently than DaCld, and whose function is currently unknown; and Staphylococcus aureus Cld (SaCld) also known as HemQ, which has no chlorite decomposing activity, but is essential for heme biosynthesis. In addition to understanding of how the active site environment variables direct the reactivities the three Cld types, we expect to gain insight into 1) a novel mechanism of O2 production, 2) the possible role(s) of Clds in Gram-negative pathogens like K. pneumoniae and 3) a new pathway in heme biosynthesis in critically important Gram-positive pathogens like S. aureus, whose drug-resistant strains are plaguing healthcare facilities throughout the US. As no members of the Cld family from Gram-positive bacteria are found in humans, Cld holds promise as a yet unexploited target for antimicrobial therapeutics, once the mechanistic aspects of their functions are understood. Specifically, the aims of the project are threefold: 1) elucidat structural characteristics of intermediates key to O2 evolution in the DaCld/chlorite-decomposing reaction, 2) examine enzyme reactivity and reaction intermediates of KpCld with chlorite and peroxynitrite to assess possible detoxification function(s) of KpCld, and 3) parameterize role of SaCld (HemQ) in heme biosynthesis by determining its reaction mechanism with coproheme. These aims will be addressed with spectroscopic (resonance Raman and transient absorbance) and kinetic (stopped flow and freeze-quench) approaches to determining atom connectivities and structures and electronic properties of Cld reaction intermediates. These studies support our long term goal of understanding how heme environment directs enzyme function.